The selective degradation of intracellular proteins is of central importance for the generation, function and survival of eukaryotic cells. The ubiquitin-proteasome system (UPS) is responsible for the controlled degradation of most intracellular proteins, and abnormal regulation of the UPS is associated with a variety of human diseases, including cancer, myopathies, and neurodegenerative disorders. Although dramatic progress has been made in understanding the structure and function of proteasomes, we still know extremely little about how proteasome activity is dynamically regulated in time and space. The activity of the 26S proteasome declines with age, but the underlying molecular mechanisms remain unknown. Our prior work focused on the regulation of caspases by the UPS, and results obtained during the current funding cycle revealed the joint use of proteasomes and caspases in the controlled demolition of cellular structures that is needed for terminal sperm differentiatio in Drosophila. Similar mechanisms are thought to mediate the remodeling of other cell types, including neurons and muscle, in both insects and vertebrates. The overall goal of this project is to understand how proteasomes are regulated to promote changes in the cyto architecture, size and survival of cells, and how this process affects age-related neuronal degeneration. We recently discovered a novel proteasome regulatory mechanism that offers unique opportunities to study proteasome regulation in the context of both normal organismal development, and in response to stress and aging. In particular, we found that the ADP-ribosyl transferase Tankyrase (TNKS) binds to and critically activates the proteasome regulator PI31 (Proteasome Inhibitor of 31kDa) to promote 26S proteasome assembly. These results suggest a potential mechanistic link between energy metabolism, NAD+, DNA-damage and proteasome regulation that is likely to play important roles in development, protein homeostasis and aging. Here, we will investigate the biological role and regulation of TNKS/PI31-mediated proteasome activation. We propose to use a multi-disciplinary approach that integrates Drosophila genetics, developmental biology, cell biology, and neurobiology, biochemistry, and small- molecule chemical inhibitors. Amongst other things, we will test the specific hypotheses that the TNKS/PI31-pathway is regulated by NAD+ that activation of this pathway protects against phototoxic stress and that diminished activity of this pathway with age causes increased vulnerability to neuronal degeneration. The current proposal brings, for the first time, the full power of Drosophila genetics and molecular biology to investigate these questions and combines it with biochemical studies in both insect and mammalian cells to explore new paths towards the clinic. We expect that this project will fundamentally advance our understanding of how protein degradation is regulated and provide new insights how to manipulate this process for the treatment of human diseases.